3.4 Preparation and single molecule investigation of highly structured do-
3.4.1 Growth of the highly structured domains
3.4.1.1 Introduction
An important finding of this study is that CTAB-templated mesoporous films with small domain sizes, such as those investigated above, can be treated after synthesis to lead to more structured materials with macroscopic-sized domains of nearly perfectly linear channel systems. A critical parameter of the post-synthetic treatment of the mesoporous films is the relative humidity (RH). It is known that water plays a key role during and after the synthesis of the mesoporous films.101–105 First, water is essential in the fabrication of these films to ensure the hydrolysis of the silica precursor. Moreover, the structure of the silica matrix with template filled pores is fully rigid in nature, and resembles partially a liquid crystal. This flexibility allows rearrangement of the silica structure even after the synthesis of the film and the evaporation of the solvent. RH plays an important role in this phenomenon because water molecules can be found at the
surface of the micelles as well as in the silica network itself. The influence of water on the porous structure of CTAB-templated systems has been investigated in details by Grosso et al.101, 102, 144 and Gibaud et al.103–105 An interesting result of these studies is that raising the RH can yield phase transformations after synthesis of the film, which clearly shows that strong rearrangements of the mesoporous material can occur. However, the timescales on which these experiments were conducted didn’t exceed some hours after the synthesis of the mesoporous films. Indeed, after this period of time the mesoporous films were considered to have reached thermodynamical equilibrium because no significant changes could be observed on longer timescales with the methods of analysis used in other studies.
Mesoporous materials are commonly characterized by X-Ray diffraction (XRD) and transmission electron microscopy (TEM).40, 41 However, these standard techniques have severe limitations. From XRD patterns the average pore-to-pore distances in the host-system can be calculated, but no information about the alignment of the pores on the substrate and on the arrangement of domains of parallel pores can be obtained. In contrast TEM gives a very detailed image of the pore structure, but the areas of the sample which can be imaged are limited to a few hundreds of nanometers in size, and more importantly TEM imaging requires a special, mostly invasive preparation, like scratching, grinding and ion milling.
The possibility to identify easily these large domains by the fluorescence signal emitted from guest dye molecules such as TDI incorporated into the channels, and to measure areas of the mesoporous films on length scales up to hundreds of micrometers makes fluorescence microscopy an ideal tool to investigate the formation and the growth of large highly structured domains of linear channels within a mesoporous film.
3.4.1.2 Fabrication of large highly structured domains
TDI dye molecules were incorporated in the pores of CTAB-templated mesoporous films (Synthesis procedure described in section 3.5). However, here the fluorophore is used at ensemble concentration (c = 10−6 mol/L) in order to render visible the totality of the domains investigated. Additionally, a lambda half plate is constantly and slowly rotated during the scan of the laser beam in order to measure the orientation of the dipole moment of the dye molecules.
Figure 3.15a shows a patchwork of nine fluorescence images recorded from a region of about 500 µm in total size of a CTAB-templated mesoporous film, and taken directly after spin-coating of the film (time 0). An homogenous fluorescence signal is visible over the whole fluorescence image. In particular, no modulation of the fluorescence intensity
Figure 3.15: Slow formation of highly structured domains after spin-coating of the CTAB-templated mesoporous films. (a) Patchwork of nine confocal fluorescence images of a mesoporous film loaded with TDI at ensemble concentration observed directly after spin-coating. Only a fluorescence background signal is visible. (b) Same sample after 15 days of storage at a constant RH of about 50 %. Bright, highly structured domains have been formed. (c) Another sample after 15 days of storage at a constant RH of 50 %. The form of the bright domain is different.
can be detected during the revolution of the polarization of the laser. This indicates that the TDI molecules are distributed equally over the sample without preferential orientation of their dipole moments. This is consistent with the TEM micrographs shown in Figure 3.6a and b. Indeed, the small size (10 - 100 nm) of these regions and the curvature of the channels explain that no preferential orientation of the TDI dye molecules is expected on the lengthscale of the optical resolution of a confocal microscope, which is about 300 nm.
Figure 3.15b shows a patchwork of nine fluorescence images of the same sample 15 days after synthesis. During this period, the sample was stored at a constant RH of about
50%. In addition to the homogeneous fluorescence background, regions up to 100µm in size exhibit a much brighter fluorescence signal. The formation of such regions is observed in all the ten investigated samples stored at the same RH. However, it is observed that the shape and the size of these regions can vary. Figure 3.15c corresponds to another sample synthesized and stored at the same experimental conditions. Here, the domains exhibit a more rectangular shape, and are slightly smaller in size (tens of micrometers).
Figure 3.16a shows a zoom in such a region. Stripped horizontal lines, with the same periodicity in time as the polarization modulation of the laser beam, can be seen over all the three distinct regions. The fact that the fluorescence signal is modulated and demonstrates that TDI dye molecules have a preferential orientation in each of these re-gions. Indeed, an homogeneous signal would result from randomly oriented fluorophores which emit light in all directions. Moreover, the diffusional and orientational behavior of the TDI dye molecules in the channels of these regions will be investigated in section 3.3.2. As will be shown, the molecules show linear movement, with their transition dipole moment aligned along their trajectories, reflecting well-ordered structural areas of the host. Hence, it can be concluded that these regions of bright, oriented dye molecules correspond to highly structured domains of parallel channels in which the fluorophores are remarkably aligned along the direction of the pores. Moreover, the fact that all the dipole moments of the TDI molecules lie in these domains perfectly perpendicular to the optical axis of the setup offers an ideal configuration for an optimal excitation of the fluorophores. This can explain the much brighter signal observed for TDI molecules encapsulated in such domains compared to the rest of the mesoporous film.
An additional important information that can be obtained from the analysis of these polarization-modulated patterns is the absolute direction of the channel system in the structured domains. The insert shown in Figure 3.16a is a zoom in the region highlighted in red. The junction between two domains can be clearly seen in the middle part of the image (indicated by an arrow). Two domains have grown until they have reached each other. A phase shift in the stripes of the polarized fluorescence light can be clearly seen at the junction of the two domains, revealing two distinct directions of the channel system.
The analysis method of such polarization-modulated fluorescence images is explained in detail in Section 2.1.3.4. Fits of the fluorescence patterns were computed at arbitrary and equally distributed positions in the domains. The orientation of the molecules and thus of the pores at these positions are overlayed to the fluorescence image as yellow bars in Figure 3.16b. The angle between the pore directions at the junction of these two distinct domains is about 50◦. A very interesting observation is that the channels are not perfectly parallel over a lengthscale of tens of micrometers inside a domain, but can have a certain curvature. This can be observed for each of the three domains shown in 3.16b. The pores are slightly curved like in the domain situated in the left upper part
Figure 3.16: Polarization modulated confocal fluorescence images of three highly structured domains.
(a) The stripes in the domains indicate that the TDI molecules are oriented in the same direction in each of the domains. The inset shows a zoom into the region marked with the red rectangle. A phase shift at the boundary of the two domains shows clearly that the orientation of the fluorophores in each domain is different. (b) The same fluorescence image with orientations of the dipole moment of the TDI molecules overlayed as yellow bars at different arbitrary positions (see section 2.1.3.4 for details about the analysis of the data) . The orientations shows that the pores are not perfectly parallel within a domain, but slightly curved over a lengthscale of tens of micrometers.
of the image, or the direction of the channels can show a roughly circular symmetry, growing in all directions as can be seen for the two remaining domains of the image.
3.4.1.3 Influence of the relative humidity
We have seen that these highly structured domains are not present in the samples directly after the synthesis of the films, but are formed on timescale of days after rearrangement of the mesoporous network, which leads to higher ordering of the system with pores oriented over a macroscopic lengthscale. The formation and the growth of these domains is a very slow process and is strongly dependent of the RH in the atmosphere surrounding the sample during its storage. The influence of the RH was investigated by recording at different time points confocal images of six samples synthesized at the same time point, and under the same experimental conditions. The samples were stored directly after synthesis at different RH: 0%, 30%, 40%, 50%, 60% and 80%.
Figure 3.17 shows patchworks of confocal images for these six samples after 15 days of storage. The first sample (0% RH) was placed in a desiccator in presence of silicagel to ensure the absence of water in the atmosphere surrounding the sample. Only fluorescence background is visible for this sample (Figure 3.17a). In contrast, the fluorescence images of the sample stored at 30% RH (Figure 3.17b) show the presence of many small domains (about 1µm in size). Oriented domains can also be observed for the two samples at 40%
RH and 50% RH (Figure 3.17c and d respectively). However, they are much larger in size (up to 100 µm) than for the sample stored at 30% RH.
Surprisingly, no domains can be observed for the two last samples stored at 60% RH and 80% RH. Instead, small crystal-like particles are visible (Figure 3.17e and f). However, their shape is very irregular and their fluorescence signal shows no modulation under rotation of the polarization of the laser beam. It is known that CTAB-templated films degrade in presence of water. The presence of the small particles indicates that the mesoporous films are unstable above a RH of 60%. To confirm the destructive effect of high RH on the samples, Atomic Force Microscopy (AFM) measurements were performed of the surface of the mesoporous films. A region of 40 µm × 40 µm of the surface of a film stored during 15 days at 40% RH after synthesis was imaged (Figure 3.18a).
An immediate observation is that the surface of the film is not uniform as would be expected from the last layer of pores on the top of the film. In fact, many holes (identified in the AFM image by dark signals) can be seen at the surface of the sample. Figure 3.18b shows a cross-section along the red line displayed in the AFM image. The graph exhibits steps of 3 - 4 nm in height. This value corresponds roughly to the layer-to-layer distance that was calculated from the XRD patterns (Figure 3.20c). It can be concluded that the layers at the top of the film are not perfect. Hence, in this image at least the two first different layers of pores can be distinguished.
Figure 3.17: Influence of the relative humidity (RH) on the growth of the domains. Patchwork of nine confocal fluorescence images of a mesoporous film loaded with TDI at ensemble concentration observed directly after 15 days of storage at a constant RH of about (a) 0%, (b) 30%, (c) 40%, (d) 50%, (e) 65%, and (f) 80%. The optimal range for the growth of large, highly structured domains is 40 - 50%. At very high RH the films degrade.
Figure 3.18c shows an AFM image of the same sample after 1 h of storage at 80% RH.
This image is dramatically different from the previous one. The cross section displayed in Figure 3.18d shows that some plateaus separated by about 4 nm are still visible. However, their surface is much rougher than in Figure 3.18b, and shows some granularity. This demonstrates that water has an invasive action on the CTAB-templated mesoporous films. Considering that the sample was stored only one hour at 80% RH, it is very probable that the two samples stored at 60% and 80% RH described previously were degraded by water.
To conclude, it has been shown that the RH of the atmosphere in which the mesoporous films are placed during storage plays a critical role in the formation of the highly struc-tured domains. No domains were formed if water was suppressed from the surrounding atmosphere, and the optimal range for the growth of large domain is around 40% - 50%
RH. Below this value, the domains do not appear or are relatively small (in the order of micrometer). Above this value the mesoporous film degrades under the presence of excessive water.
Figure 3.18: AFM images of the surface of a film before and after storage at high RH. (a) AFM image directly after spin-coating of a film. (b) Cross-section along the red line shown in (a). Plateaus can be observed separated by about 3 - 4 nm, corresponding the 2 -3 last layers of pores. (c) The same sample after 1 h of storage at 80 % RH. (d) Cross-section along the red line shown in (c). The plateaus are nearly not visible anymore, indicating degradation of the film.
3.4.1.4 Time-dependent measurements of the growth of the structured do-mains
The phenomenon of formation of highly structured domains days after the synthesis of the mesoporous films opens new questions: what is the starting point of the growth of the domains? What are the kinetics involved? Is the reaction of formation of these domains over after a certain period of time, or can the domains grow as long as the experimental conditions are maintained constant?
To address these questions the same area of a TDI-loaded mesoporous film was imaged by confocal microscopy during a period of 15 days after the synthesis. The sample was kept at a constant RH of about 45% during the observation time, and times series of fluorescence images were recorded. The time interval between two frames is 45 min, and the polarization of the laser was kept linear here. Figure 3.19a shows a fluorescence image of the mesoporous film recorded 5 days after spin-coating. The formation of the structured domains has still not started at this point in time, which is the starting point of the measurements (time 0). The fluorescent images of this area taken during the next 21 hours (data not shown) are identical to Figure 3.19a i.e. no highly structured domains are observed. However, after 21 h of observation time, a first fluorescent spot appears
(Figure 3.19b), indicating the abrupt starting point of the formation of structured do-mains. Figures 3.19c shows the image of the same region at t = 22.5 h. One can observe that the domain corresponding to the fluorescent spot of Figures 3.19b has dramatically grown, and that two additional domains appeared. It is very interesting here to note that the formation of each of the three domains start almost exactly at the same time (within 1 h 30 min).
The growth of these domains can be followed in the next frames. Figures 3.19d and e show the same domains at t = 24 h and 54 h, respectively. An immediate observation is that the size of domains clearly increases with time. This growth can be quantified by plotting the area of a domain as a function of time. The area of the domains was measured using a threshold for the fluorescence intensity in the confocal images. Figure 3.19e shows such a threshold image corresponding to t= 24 h. The domains appear here as white pixels whereas the background is black. The area of the domain to the left is plotted versus time in Figure 3.19f. As mentioned above,the domain does not exists in the first 21 h of measurement time. It appears at t = 21 h, and its total area increases until it reaches a maximum at about t = 40 h. After this time the silica matrix has probably lost much of its flexibility, so that the rearrangement of the structure is not possible any longer.
The fact that the domains appear suddenly more than 5 days after synthesis of the meso-porous film is surprising, and suggests that a factor external to the system (for example a nimble movement of the setup) initiated their formation. This phenomenon is an ag-ing process which is equivalent to crystal growth where the apparition of micro-crystals initiates the whole process of crystalline growth. Six other samples investigated under the same experimental conditions showed a similar behavior i.e. sudden appearance of highly structured domains some days after the synthesis. However, the time lag before apparition of the domains can vary between 3 and 9 days depending on the sample.
The need for a certain time lag before formation of the domains may indicate that the silica matrix has to achieve enough condensation before reorganizing into a more stable structure. In a too early stage after synthesis, the system is probably still too flexible, and would probably not gain in stability by rearranging its pore structure. On a longer timescale (tens of days up to months), however, nearly no modification of the domain areas is observed (data not shown). An explanation is that the silica structure is fully condensed after such a long time, so that the whole system becomes too rigid to allow reorganization of its matrix. Hence, the phenomenon of formation of highly structured domains seems to be very sensitive to the condensation process of the silica: there is an optimal degree of the silica condensation where the mesoporous film gains in stability by reorganizing its structure.
Figure 3.19: Real time measurements of the growth of highly structured domains in a CTAB-templated mesoporous film loaded with TDI at ensemble concentration. (a) Confocal fluorescence image of the film 5 days after spin-coating which is the time zero for these measurements. Only a fluorescence background is detected. (b) The same region of the mesoporous film observed after 21 h. A first domain appears. (c) After 22.5 h. Two other domains are visible. (d) and (e) The same domains after 24 h and 54 h respectively. (f) Threshold image of the image shown in (d) used for the analysis of the area of the domains. (g) Plot of the total area of the domain to the left versus time. The formation of the domains starts suddenly after 21 h of observation time, and a maximum of the area of the domains is reached about 20 h later.
3.4.1.5 Conclusion
It has been shown that the mesoporous structure of CTAB-templated thin films can rearrange on a timescale of days after synthesis into large, highly structured domains of
extremely linear channels. The formation and the growth of these domains is strongly dependent on the RH, with an optimal range of 40 - 50% of RH for the formation of large domains (up to 100µm in size). The growth starts suddenly after a storage period of 3 - 9 days, and the area of the domains reaches their maximum several hours later.
Further investigations with complementary methods such as X-ray scattering or TEM are currently in progress in order to characterize and to understand the phenomenon of the formation and growth of these highly structured domains in depth.